Detection of Ligand-binding to Membrane Proteins by Capacitance Measurements.

In multi-cellular organisms, cells communicate with each other utilizing chemical messengers. For many of these messenger molecules, the membrane is an insurmountable barrier. Yet, they act by binding to surface proteins often triggering a cascade of reactions inside the cell. Accordingly, studying ligand-receptor interactions at the cellular surface is key to understanding important aspects of membrane biology. However, despite a multitude of approaches to study membrane features, there is a need for developing techniques that can measure ligand binding with high temporal resolution and on a single cellular level. We recently developed a label-free approach to study ligand binding in real time. This methodology capitalizes on changes of the membrane's surface potential induced by the adsorption of a charged ligand. The resulting apparent alteration of membrane capacitance is measurable by capacitance recordings. Herein, we describe the implementation of the same using recordings obtained from HEK293 cells stably expressing the human serotonin transporter (SERT), which were challenged with the inhibitor cocaine.

2. Split cells, at 80% confluency. Detach them with PBS/EDTA and spin them down at 80 x g for 5 Copyright  3. Induce the expression of GFP-SERT in Tet-on HEK293 cells with tetracycline, twenty-four hours prior to the experiment. Plate cells at low density (2,000-4,000 cells ml -1 ) onto PDL-coated 35 mm dishes containing 2 ml maintenance medium supplied with 1 μg•ml -1 tetracycline.
C. Cleaning of the perfusion system and positioning of the perfusion manifold tip 1. Clean the Octaflow perfusion system and manifold using 1% tergazyme (10 g tergazyme dissolved in 1 L MilliQ-water; prewarmed to 37 °C, incubation time 30 min) followed by 7-10 wash steps with MilliQ-water.
2. Fill the Octaflow tanks with extracellular solution and connect them with the manifold tubing.
Ensure that no air bubbles are trapped within the tubing.
3. For the positioning of the manifold tip, immerse the manifold tip into deionized water and start applying extracellular solution using a pressure of 400 mmHg.       The impulse response must be re-recorded, if changes to the apparatus are made that affect the signal path, the sampling frequency or the corner frequency of the low-pass filter (i.e., replacement of the amplifier, digitizer (Digidata 1440) (BNC-cables etc.).
1. Mount a 10 MΩ resistor between the signal input and the ground input of the head-stage. Run the protocol to measure the impulse response function (Step E1, Figure 6A).
2. Record the stray capacitance of the 10 MΩ resistor. For this, disconnect the 10 MΩ resistor from the ground input while keeping the resistor connected to the signal input of the head-stage and rerun the protocol to measure the impulse response function (Step E1, Figure 6B). Copyright   Note: Once adjusted, do not change it during the experiment. 6. In most cases, it is not possible to fully compensate the stray capacitance with the compensation circuitry. We therefore recommend recording the remaining stray capacitance of the pipette and the membrane patch beneath it, using the protocol described in Step E2. The recorded currents can later be subtracted from the membrane currents obtained with the protocol to measure the membrane capacitance of the whole cell (Step E3).

Data analysis
MatMecas is a Matlab-based software, which is used to estimate the three parameters of the minimal equivalent circuit of a cell. They are: CM-membrane capacitance, RM-membrane resistance and RA-access resistance ( Figure 7A). For these estimations, the recorded currents elicited by a train of bipolar square-wave voltage pulses are de-convolved with the impulse response function of the recording apparatus. This procedure reconstructs the initial segment of the current decay, which is distorted by intrinsic filters in the circuitry of the amplifier ( Figure 7B). The reconstructed decays are then fit to a monoexponential function ( Figure 7C). The obtained fit parameters (IP, IS and τ) are used to estimate circuit parameters of the minimal equivalent circuit by utilizing the equations shown in Figure 7D. The detailed description of the analysis can be found in (Hotka and Zahradnik, 2017). MatMecas reads currents stored in axon binary files (abf) as well as txt files. The installer application will take the user through the installation process and will download all the necessary files including MCR. Thus, it is necessary to install MatMecas on a computer connected to the internet. To ensure availability of the required permissions we recommend running MatMecas and the installer app as an administrator.
B. MatMecas user interface description (Figure 8) For description see below: